Proteomic Studies in Human Genetic Disorders. Several studies have been performed by the facility in collaboration with NICHD IRP clinical groups to determine changes in protein expression in several animal models of human genetic disorders or for tissues from patients with specific genetic disorders. In these studies, changes in protein expression were quantified by the intensity of spot staining for proteins separated by 2-dimentional gel electrophoresis (2D-GE). Differentially expressed proteins were then identified by in-gel digestion and peptide analysis by MS/MS fragmentation. Studies using this approach have been recently completed for two mouse models of human diseases: a) Niemann-Pick Disease-Type C1 (NPC1), and b) tumor aggressiveness of pheochromocytomas/paragangliomas. For the NPC1 mouse model, Npc1 knock-out and wild type mice were compared, and 77 differentially expressed proteins were identified from mouse cerebellum. Translating these findings from the animal model to CSF from NPC1 patients, three proteins with altered expression in the animal model (glutathione-S-transferase a, superoxide dismutase, and FABP3), were also found to have altered expression in CSF from NPC patients. Currently, this approach is being applied to identify changes in protein expression in adrenal gland tissue from patients with a variety of adrenal gland disorders. Identification of LECT2-associated Amyloidosis in Adrenal Tissue. We recently identified leukocyte cell-derived chemotaxin-2(LECT2) as a component in the formation of amyloid plaques in adrenal tissue. Although adrenal tissue was positive for amyloid by Congo Red staining, specific immunostaining for proteins commonly known to form amyloid plaques were all negative. Plaque proteins from disease tissue were extracted and separated by1D SDS/PAGE. After digestion of the gel bands, serum amyloid P-component and leukocyte cell-derived chemotaxin-2 (LECT2) were identified as components of these plaques. LECT2 has been reported to be found in amyloid plaques in kidney, but this is the first observation of this protein causing amyloidosis in adrenal tissue. The high accumulation of LECT2 in adrenal amyloid plaques from this patient was confirmed by Western blots using a specific LECT2 antibody, which were positive for the disease tissue, while negative for comparable amounts of tissue from normal adrenal glands. Protein Profiling and Quantification in Cerebral Spinal Fluid (CSF) for Disease Biomarkers. Two complementary approaches have been applied to characterize protein biomarkers in CSF from patients with NPC. The first approach uses a novel analytical method that we developed for the collection of MALDI TOF (Matrix Assisted Laser Desorption Ionization-Time Of Flight) mass spectrometry data and analysis of the spectra by the combined use of ANalysis Of Variance coupled with Principal Component Analysis (ANOVA-PCA). This method has detected significant changes in a number of relatively low molecular weight proteins and peptides in CSF, and further work is in progress to identify the specific proteins/peptides. We have also applied a second, more global, approach to quantitate relative protein expression in CSF from NPC patients, using an 8-plex iTRAQ labeling process to mass tag peptides generated from proteins present in each sample. This method is designed to perform relative protein quantification while maintaining individual patient information and providing the ability to compare results across multiple iTRAQ experiments. Initial studies using the Npc1 knock-out mouse model have been completed to determine the analytical variation of this method. Several differentially expressed proteins identified in this pilot study were also identified in our previous study of differentially expressed proteins in the cerebella of via 2D-GE. This method is now being applied to CSF from patients with the fatal, neurodegenerative, Niemann-Pick Disease, type C1 in order to profile protein changes and identify biochemical alterations correlated to disease progression and treatments. Characterization and Quantification of Serum Cardiolipins. We have developed a methodology to extract and quantify cardiolipins in human serum. Cardiolipins (CLs) are a major lipid in mitochondrial membranes. However, CLs can also be detected in serum, and their metabolism in serum has not been previously characterized. The analytical approach involves addition an internal standard to a serum sample, followed by liquid-liquid and solid phase extractions. Cardiolipins are then detected and quantified by LC-MS analysis. Using this method, we have analyzed cardiolipins in normal adult serum, plasma, and whole blood. The major CL in serum was identified as (18:2)-4 cardiolipin, in a concentration range between 3 -10 nM, approximately 1000-fold lower than reported by earlier, less accurate measurements. In addition, cardiolipins with other fatty acid moieties have been detected in serum using this approach, and the fatty acid composition of serum CLs was distinct from the composition found in plasma or whole blood. This method is currently being applied to quantify cardiolipins in maternal serum, cord blood, and infant serum from a clinical study. In some of these patient samples, a family of novel CLs have been identified and appears to be associated with bacterial infections and antibiotic treatment. Mass Spectrometric-Based Profiling of Urinary Steroids. Current approaches to the analysis of urinary steroids typically employ either immunoassay or mass spectrometry based technologies. Immunoassay-based methods often lack specificity due to cross-reactivity with other steroids, while targeted LC-MS/MS is limited to the analysis of pre-determined analytes. We have developed a new LC-MS/MS approach to urinary steroid profiling that enables us to detect the steroids that have truly changed in a patient cohort without knowing their identity beforehand (i.e., untargeted metabolomics of steroids). In addition, we have developed a product ion spectrum database of known steroids to improve our capability to identify novel steroids. These methods have been applied to a pilot project to investigate urinary steroids for patients diagnosed with polycystic ovarian syndrome (PCOS). Initially these studies detected elevated levels of an unknown compound consistent with an androgenic steroid in PCOS patients. We were then able to identify the unknown as a mixture of androsterone-sulfate and etiocholanolone-sulfate. A two-dimensional liquid chromatographic method, using C18 in the first dimension and Porous Graphitic Carbon in the second, was applied to separate the diastereomeric pair for quantification. This approach is being further extended to additional PCOS patients and to studies on patients with Congenital Adrenal Hyperplasia (CAH).